Project Summary A detailed understanding of human health and disease requires methods to probe cellular behaviors as they occur within intact organ structures and living subjects. In recent years, technologies have emerged from the imaging community that enable diverse biological features to be visualized and tracked in real time. While powerful, these approaches have been largely confined to monitoring cellular behaviors on a microscopic level. Visualizing cellular functions across larger spatial scales?including those involved in cancer progression and migration?requires new imaging tools. Grant GM107630 aims to develop general strategies for macroscopic, multi-cell tracking with bioluminescent tools in living organisms. Bioluminescence imaging is a powerful technique for visualizing small numbers of cells in rodent models. This technology employs enzymes (luciferases) that produce light upon incubation with small molecule substrates (luciferins). Several luciferase-luciferin pairs exist in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for in vivo imaging use the same substrate, and therefore cannot be used to distinguish multiple cell types in a single subject. The PI and her team have demonstrated that the substrate-binding interface of firefly luciferase can be re-engineered to generate panels of mutant enzymes that accept chemically distinct luciferins. When mutants and analogs are mixed together, light emission is produced only when complementary enzyme-substrate partners interact. Ongoing work seeks to generate improved orthogonal imaging tools using a combination of rational design and screening. Our goals include (1) uncovering the molecular determinants of orthogonality (via crystallography and deep-sequencing analyses) for lead pair optimization; 2) generating orthogonal probes with improved tissue penetrance; and 3) imaging tumor heterogeneity with expanded orthogonal toolsets. Our research would be dramatically accelerated by the purchase of an all-in-one fluorescence microscope. Successful translation of the orthogonal luciferases in vivo requires benchmarking the bioluminescent enzymes against established markers (e.g., fluorescent proteins). Luciferase-fluorescent protein constructs also provide a ?one-stop-shop? for tool users, enabling macro-scale imaging and analyses (via bioluminescence) and micro-scale imaging/ex vivo analyses (via fluorescence). The fluorescence microscope will also enable the development of probes that can demarcate cellular location and function within heterogeneous systems. Such tools will further augment our studies of tumor-immune cell interactions. The proposed research is significant, as the imaging tools will enable the direct interrogation of cellular networks not currently possible with existing toolsets. Such studies may fundamentally change existing views on cancer progression. Additionally, similar to other imaging technologies, the probes will likely inspire new discoveries in a broad spectrum of fields.